In broad terms, analytical chemistry involves some combination of separation, identification, and quantitation of analytes of particular interest. To accomplish these goals, it is necessary to provide a sufficient concentration of the analytes at the detection point in the analytical device to register a signal at the detector that is distinguishable from background signal. With analyte concentrations below parts per million (ppm), many analytical detection systems are inadequate to detect the analyte.
Capillary electrophoresis (CE) is a high-resolution technique for separating charged analytes in liquid solutions using electric fields. The micron-scale radial dimensions of the capillary serve to dissipate Joule heating and control convection efficiently at high separation voltages, allowing for plate heights of micrometers or less. However, because of the small radial dimension of a capillary, it is difficult to detect short injected plug lengths of low-concentration analytes using standard optical detection set-ups (e.g., UV-VIS absorbance detection). Hence, the injection of large sample volumes (i.e., long sample plugs) has been necessary to improve the detection of analytes present at low concentration. Normally, the injected length of an analyte plug injected into some form of separation column, determines the minimum peak width at the detection point. As a result of the fact that separation columns are of a finite length, injection plug length is limited, in practical terms, to the millimeter range—beyond this, the high-resolution capabilities of capillary electrophoresis cannot be preserved. However, this problem has been circumvented by protocols that invoke mechanisms that narrow, or stack, much longer analyte zones into sample plugs. Stacking concentrates the analytes into smaller space, decreasing the length of an analyte zone and, thus, increasing the analyte concentration and its signal at the detector.
FIG. 8 shows an exemplary setup that can be used to practice electrophoresis. Electrophoresis unit 1 is interfaced with data collection/storage unit 14, which contains software/firmware/hardware for control of the instruments and data collection (e.g., processor, personal computer, display monitor and printer, or personal digital assistant (PDA) or the like. Separation channel 2 (shown here as a capillary) has an entrance (inlet) end 4, an exit (outlet) end 6, and a detection window 8. Separation channel 2 is flushed by high pressure or vacuum with three to five capillary volumes of buffer. The outlet reservoir 10 is filled with buffer and holds the exit end 6 of separation channel 2. Inlet reservoir 12 is also filled with buffer and holds the entrance end 4 of separation channel 2. Sample reservoir 11 contains sample for injecting into the separation channel 2. The high voltage power unit 16 has an anode 18 and a cathode 20. Outlet reservoir 10 also holds cathode 20, while inlet reservoir 12 also holds anode 18. An electric field is then applied by high voltage power unit 16 across separation channel 2 from anode 18 to cathode 20, which causes buffer in inlet reservoir 12 to travel through separation channel 2 and into outlet reservoir 10, for electrophoretic conditioning of separation channel 2. After separation, channel 2 has been electrophoretically conditioned, anode 18 and entrance end 4 of separation channel 2 are placed within sample reservoir 11. An electric field may be applied across separation channel 2 for electrokinetic injection (injection by electrophoresis instead of by pressure) of a sample plug into separation channel 2. The electric field is applied across separation channel 2 for a period of time sufficient to permit injection of a sample plug of desired length. Alternatively, the plug could be injected into separation channel 2 using a pressure technique known in the art.
Once an analytes zone is located at the detection window 8, radiation, for example from an ultraviolet (UV) light source 34, shines through on the detection window 8 and, therefore, through the analytes—the amount of light transmitted through the sample and window is detected by the detector 36 located on the opposite side of detector window 8 from UV source 34. The detector 36 is interfaced with a data collection/storage unit 14 to collect the separation data including the number of analytes zones to traverse the window as well as the relative amount of light that was incident on the detector (which translates to “concentration”).
FIG. 9 is an illustrative setup used to practice electrophoresis. The electrophoretic unit 101 is a T-configuration cross-channel injection microchip. Separation Channel 102 has an entrance end 104, an exit end 106. Outlet reservoir 110 is filled with buffer and is connected to exit end 106 of separation channel 102. Inlet reservoir 112 is also filled with buffer and is connected to entrance end 104 of separation channel 112. Sample channel 140 has an entrance end 142 and an exit end 144. Sample reservoir 111 contains sample for injecting into separation channel 102 and is connected to entrance end 142 of sample channel 140. Waste reservoir 146 is filled with buffer and is connected to exit end 144 of sample channel 140. Sample channel 140 is configured to connect through and cross over separation channel 102 to form a ‘T-configuration’. High voltage power unit 116 has an anode connected to inlet reservoir 104 and an anode connected to sample reservoir 111, and a cathode connected to outlet reservoir 110 and a cathode connected to waste reservoir 146. An electric field is applied across separation channel 102 for electrophoretic conditioning. The field is applied from anode connected to inlet reservoir 112 to cathode connected to outlet reservoir 106. For electrokinetic injection of a sample plug into separation channel 102 (not by pressure which is the current state-of-the-art), an electric field is applied from anode connected to sample reservoir 111 to cathode connected to outlet reservoir 106 for a defined period of time. An electric field is then applied across separation channel 102 causing separation and detection of the various analyte zones. The field is applied from anode connected to inlet reservoir 112 to cathode connected to outlet reservoir 106.
To separate neutral analytes by CE, it is necessary to provide an electrokinetic vector. The first example of this, micellar electrokinetic chromatography, utilized a charged micelle to impart mobility to neutral analytes. As a result of the successful use of other electrokinetic vectors that are not micellar in character, (e.g., charged cyclodextrins) electrokinetic chromatography (EKC) is a term that has been utilized to encompass the use of any electrokinetic vector for separation of neutral analytes. Recent techniques for stacking neutral analytes in EKC require pressure injections of sample matrixes. For instance, high-salt stacking and sweeping have been applied to afford sample plug lengths up to 60% of the effective capillary length (length from the injection end to the detection point). High-salt stacking utilizes discontinuous buffer conditions (the separation matrix co-ion is different from the sample matrix co-ion). The sample matrix co-ion (the ion with the same charge as the electrokinetic vector, e.g., chloride) must have a higher intrinsic electrophoretic mobility than the electrokinetic vector, and the sample co-ion must be present at a higher concentration in the sample matrix than that of the electrokinetic vector in the separation buffer.
Also, sweeping utilizes continuous sample matrix/separation buffer conditions, i.e., there are no mobility differences between the background electrolyte or buffer, between the sample matrix and the separation buffer.
In addition, pressure injections are typically low-velocity to diminish mixing at the sample matrix/separation buffer interface and to maintain reproducibility between analyses. While typical separations take as little as 60 seconds, the time necessary to introduce long sample plugs (e.g., 50% of the capillary length, ca. 50–2000 seconds) by low pressure can, in certain instances, exceed the analysis time by more than an order of magnitude.
Another drawback associated with large sample plug pressure injections is that the effective capillary length available for separation is reduced, having undesirable effects on the ability to resolve analytes. While longer capillaries might be used to overcome this problem, the 30 kV limit associated with most CE instrumentation does not allow for the same high fields (800–1000 volts/cm) typically needed for rapid, high-resolution analyses to be applied.
In addition, longer capillary lengths are not conducive with translating stacking methods from the traditional capillary format to the microchip format.
There is, therefore, a need in the art to inject large volume sample plugs (exceeding the length of the separation column) while still retaining a maximal effective capillary length for the separation mode. The electroosmotic flow can be used to inject neutral analytes in EKC without stacking, however, a concomitant stacking of neutral analytes during electrokinetic injection by electroosmotic flow is possible—this is described herein. Furthermore, the electrokinetic injection of high-salt sample matrixes by electroosmotic flow is provided, with the stacking of neutral analytes occurring simultaneously with the injection procedure, in both high-salt stacking and sweeping modes.